Compositions and methods related to isolated endophytes

ABSTRACT

Described herein are compositions and methods related to isolated Trichoderma harzianum and strains thereof.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/340,597, filed Jul. 25, 2014, which claims the benefit under 35U.S.C. § 119(e) of U.S. provisional application Ser. No. 61/858,819,filed Jul. 26, 2013, the disclosures of each of which are incorporatedby reference herein in their entirety.

FIELD OF THE INVENTION

The disclosure relates to isolated endophytes and uses thereof, such asfor the inoculation of plants to promote stress tolerance and/orincrease growth or germination.

BACKGROUND OF THE INVENTION

Symbiosis, defined as the living together of two or more organisms inclose or intimate association, is often a mutually beneficialinteraction between two organisms. Most plants are symbiotic with fungi(Petrini, 1986) and these fungi play important roles in the structure,function, and health and adaptation of plant communities (Bacon andHill, 1996; Clay and Holah, 1999; Petrini, 1986; Read, 1999; Rodriguezand Redman, 1997).

It has been demonstrated that a symbiotic relationships with fungi canenhance the growth of host plants under stressful conditions. Forexample, specific strains of endophytic fungi confer tolerance to hostplants against extreme environmental conditions including temperature,drought, and salinity (Redman et al., 2002b; Rodriguez et al., 2004;Rodriguez and Redman, 2008; Rodriguez et al., 2009; Redman et al., 2011;Rodriguez et al., 2012). Endophytes are a class of fungal symbionts thatreside within host plant roots, stems and/or leaves. In addition topromoting stress tolerance, endophytes also increase nutrientacquisition and growth rates (biomass and yields) of host plants, andimprove water use efficiency (Rodriguez et al., 2008; Rodriguez et al.,2009).

Abiotic stresses (such as drought, temperature, salinity, pH andnutrient) alter the physiology of plants resulting in decreased fitness,health, development and yields. Propagating plants under marginal growthconditions (typically due to abiotic stresses) would allow for anincrease in agriculture production and mitigation of climate inducedhabitat changes for both crop and native plants. Moreover, creating newvegetation is important for soil and water remediation of polluted sitescreated by modern industry and other human activities.

Previous published studies (Redman et al., 2002b & 2011; Rodriguez etal., 2008 & 2012) have demonstrated that a special class of fungalsymbionts (class 2) can confer biotic and abiotic stress benefits toplants simply by colonizing them with the fungal endophyte.

SUMMARY OF THE INVENTION

As described herein, it has been discovered that fungal endophytes suchas Trichoderma harzianum, e.g., strain ThLm1, conferred superiorabiotic/biotic stress tolerance and increased yields of bothagricultural and native plants compared to previously identified fungalendophytes. Accordingly, aspects of the disclosure relate tocompositions and methods related to isolated fungal endophytes, such asthe species T. harzianum, including strain ThLm1.

According to one aspect of the invention, an isolated Trichodermaharzianum strain ThLm1 fungus is provided, deposited at the U.S.Department of Agriculture Culture Collection under Patent DepositDesignation number NRRL 50846 (date of deposit Jul. 26, 2013), or aprogeny or spore thereof. Compositions comprising the Trichodermaharzianum strain ThLm1 fungus and/or a progeny and/or a spore thereofalso are provided. The compositions include, for example, liquidcompositions and powder compositions.

According to another aspect of the invention, methods for promotingstress tolerance and/or enhancing plant growth or seed germination areprovided. The methods include inoculating a plant or a plant seed withthe isolated Trichoderma harzianum strain ThLm1 fungus, or a spore orprogeny thereof; or a composition thereof. In some embodiments, thestress tolerance is drought tolerance, salt tolerance, reduced nutrienttolerance, fungal tolerance, and/or temperature tolerance. In someembodiments, the enhanced plant growth or seed germination comprises anincrease in size, extent of root development, germination rate of seeds,chlorophyll content/level, photosynthetic efficiency, yield or mass.

Inoculating the plant can, in some embodiments, include colonizing aroot and/or stem of the plant with a Trichoderma harzianum strain ThLm1fungus or a spore or progeny thereof.

In some embodiments, the method further comprises growing the plant orplant seed, which may be in soil characterized by high salinity, lowmoisture, and/or low nutrient content. In some embodiments, the highsalinity is greater than 35 mM amount of a salt. In some embodiments,the low moisture content is between 0-0.18 in³ water/in³ soil. In someembodiments, the low nutrient content is fewer than 80 lbs/acre ofnutrient, wherein the nutrient comprises 20% each of nitrogen,phosphorus, and potassium, and associated micronutrients.

In some embodiments, the plant or plant seed is grown in watercharacterized by high salinity and/or low nutrient content.

In some embodiments, the plant or plant seed is grown or germinated atan average temperature at or above 35 degrees Celsius or at or below 15degrees Celsius.

In some embodiments, the plant or plant seed is a crop plant or cropplant seed, such as watermelon, tomato, corn, wheat, soybean, cucurbits,peppers, leafy greens, barley, cotton, beans, peas, tubers, berries,woody plants or rice, or a seed thereof. In other embodiments, the plantor plant seed is an ornamental, such as Rosaceae, Liliaceae, Azalea,Rhododendron, Poaceae or Chrysanthemum.

According to another aspect of the invention, plants or plant seeds areprovided that are inoculated with the isolated Trichoderma harzianumstrain ThLm1 fungus, or a spore or progeny thereof; or compositionthereof.

In some embodiments, the plant or plant seed is a crop plant or cropplant seed, such as watermelon, tomato, corn, wheat, soybean, cucurbits,peppers, leafy greens, barley, cotton, beans, peas, tubers, berries,woody plants or rice, or a seed thereof. In other embodiments, the plantor plant seed is an ornamental, such as Rosaceae, Liliaceae, Azalea,Rhododendron, Poaceae or Chrysanthemum.

According to another aspect of the invention, methods for increasingstress tolerance are provided. The methods include inoculating a plantor a plant seed with an isolated Trichoderma harzianum fungus or a sporethereof; or a composition comprising the isolated Trichoderma harzianumfungus or a spore thereof, thereby increasing stress tolerance of theinoculated plant.

In some embodiments, the stress is drought, elevated salt, reducednutrients and/or temperature stress. In some embodiments, the stress isnot the presence of polycyclic aromatic hydrocarbons, napthenic acids,or high pH.

In some embodiments, inoculating the plant comprises colonizing a root,stem and/or leaf of the plant with a Trichoderma harzianum strain fungusor a spore or progeny thereof.

In some embodiments, the method further comprises growing the plant orplant seed, which may be in soil characterized by high salinity, lowmoisture, and/or low nutrient content. In some embodiments, the highsalinity is greater than 35 mM amount of a salt. In some embodiments,the low moisture content is between 0-0.18 in³ water/in³ soil. In someembodiments, the low nutrient content is fewer than 80 lbs/acre ofnutrient, wherein the nutrient comprises 20% each of nitrogen,phosphorus, and potassium, and associated micronutrients.

In some embodiments, the plant or plant seed is grown in watercharacterized by high salinity and/or low nutrient content.

In some embodiments, the plant or plant seed is grown or germinated atan average temperature at or above 35 degrees Celsius or at or below 15degrees Celsius.

In some embodiments, the plant or plant seed is a crop plant or cropplant seed, such as watermelon, tomato, corn, wheat, soybean, cucurbits,peppers, leafy greens, barley, cotton, beans, peas, tubers, berries,woody plants or rice, or a seed thereof. In other embodiments, the plantor plant seed is an ornamental, such as Rosaceae, Liliaceae, Azalea,Rhododendron, Poaceae or Chrysanthemum.

In some embodiments, the Trichoderma harzianum strain fungus is notstrain TSTh20-1 or strain T-22.

According to another aspect of the invention, methods for increasinggermination of seeds are provided. The methods include inoculating aplant seed with an isolated Trichoderma harzianum fungus or a sporethereof; or a composition comprising the isolated Trichoderma harzianumfungus or a spore thereof, thereby increasing germination of theinoculated seeds.

In some embodiments, the plant seed is previously, concurrently and/orsubsequently treated with a fungicide and/or insecticide.

In some embodiments, the plant or plant seed is a crop plant or cropplant seed, such as watermelon, tomato, corn, wheat, soybean, cucurbits,peppers, leafy greens, barley, cotton, beans, peas, tubers, berries,woody plants or rice, or a seed thereof. In other embodiments, the plantor plant seed is an ornamental, such as Rosaceae, Liliaceae, Azalea,Rhododendron, Poaceae or Chrysanthemum.

In some embodiments, the Trichoderma harzianum strain fungus is notstrain TSTh20-1 or strain T-22.

According to another aspect of the invention, methods for reducingestablishment of fungi other than Trichoderma harzianum in a plant areprovided. The methods include inoculating a plant seed or seedling withan isolated Trichoderma harzianum fungus or a spore thereof; or acomposition comprising the isolated Trichoderma harzianum fungus or aspore thereof, thereby reducing establishment of fungi other thanTrichoderma harzianum in a plant growing from the inoculated seed orseedling.

In some embodiments, the plant or plant seed is a crop plant or cropplant seed, such as watermelon, tomato, corn, wheat, soybean, cucurbits,peppers, leafy greens, barley, cotton, beans, peas, tubers, berries,woody plants or rice, or a seed thereof. In other embodiments, the plantor plant seed is an ornamental, such as Rosaceae, Liliaceae, Azalea,Rhododendron, Poaceae or Chrysanthemum.

In some embodiments, the Trichoderma harzianum strain fungus is notstrain TSTh20-1 or strain T-22.

The details of one or more embodiments of the invention are set forth inthe description below. Other features or advantages of the presentinvention will be apparent from the following drawings and detaileddescription of several embodiments, and also from the appending claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure, which can be better understood by reference to one or moreof these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 is a bar graph showing the biomass response of young corn plantsto ThLm1. Nonsymbiotic (NS—bars on left of each pair) and Symbiotic withThLm1 (S—bars on right of each pair) corn plants (N=24) were grown forone month in a greenhouse without stress. Values on the Y-axes are wetweights in grams (g) with average weights shown.

FIG. 2 is a bar graph depicting corn growth and yield enhancementthrough symbiosis with ThLm1. Left=Nonsymbiotic (NS), Right=Symbiotic(S) with ThLm1. Values on the Y-axes are wet weights in grams (g) withaverage weights shown.

FIG. 3 is a graph showing corn growth and yield enhancement throughsymbiosis with ThLm1. Left two bars=Nonsymbiotic (NS), Right twobars=Symbiotic (S) with ThLm1. Values on the Y-axes are wet weights ingrams (g) with average weights shown.

FIG. 4 is a bar graph depicting seed germination of corn symbiotic withThLm1(S) or nonsymbiotic (NS). Left=Nonsymbiotic (NS), Right=Symbiotic(S) with ThLm1. Values on the Y-axes represent percent seed germinationwith average values shown.

FIG. 5 is a graph depicting increased chlorophyll content in symbioticcorn plants. Left=Nonsymbiotic (NS), Right=Symbiotic (S) maturegreenhouse grown corn plants in the absence of stress were measured forchlorophyll content (Y-axis, numbers are SPAD values) with averagevalues shown.

FIG. 6A is a series of bar graphs showing root and shoot weight of cornplants exposed to drought stress. Left bars of each graph=Nonsymbiotic(NS), Right bars of each graph=Symbiotic (S) with ThLm1. Values on theY-axes are wet weights in grams (g) with average weights shown.

FIG. 6B is a bar graph showing root and shoot weight of corn plantsexposed to low nutrient stress. Left bars of each graph=Nonsymbiotic(NS), Right bars of each graph=Symbiotic (S) ThLm1. Values on the Y-axesare wet weights in grams (g) with average weights shown.

FIG. 7 is a bar graph depicting the weight of corn exposed to saltstress. Left=Nonsymbiotic (NS), Right=Symbiotic (S) with ThLm1. Numberson the Y-axis are wet weight in grams with average weights shown.

FIG. 8 is a graph depicting cold stress tolerance in rice seedlings.Root biomass is depicted by the upper half of each bar. Root and shootgrowth are shown (cm). Shoot growth is depicted by the lower half ofeach bar.

FIG. 9 is a graph depicting field evaluation under drought stress. Yielddata is shown for five replicate plots containing 120 corn plants each.Plants symbiotic with ThLm1 produced an average of 85% more yield thannonsymbiotic (NS) plants.

FIG. 10 is a graph showing shoot and root biomass of corn eithernonsymbiotic (NS) or symbiotic (S) with ThLm1. Values on the Y-axes arefresh weight biomass in grams (g).

FIG. 11 is a photograph showing that symbiotic corn plants inoculatedwith ThLm1 produced more extensive root systems with more lateral roots.FIG. 11 is a representative photograph of Nonsymbiotic (NS, left) andSymbiotic (S, right) roots from plants in FIG. 10.

FIG. 12 is a bar graph showing root and shoot biomass of soybeans eithernonsymbiotic (NS) or symbiotic (S) with ThLm1. Values on the Y-axes arefresh weight biomass in grams (g).

FIG. 13 is a bar graph depicting leaf chlorophyll content of cornplants. SPAD measurements were taken which indicated relative %chlorophyll. Each of the three “S” bars represent three uniquepreparations of ThLm1.

FIG. 14 is a bar graph of SPAD measurements of soybean plants symbiotic(S, right bar) with ThLm1 or nonsymbiotic (NS, left bar). SPADmeasurements indicated the relative % of chlorophyll in plants.

FIG. 15 is a bar graph showing fruit yield of corn symbiotic withdifferent preparations of ThLm1(S) or nonsymbiotic (NS).

FIG. 16 is a series of bar graphs depicting the roots, shoots, and yieldof corn plants symbiotic with ThLm1(S, right bars in each graph) ornonsymbiotic (NS, left bars in each graph) under low nutrient stress.

FIG. 17 is a series of bar graphs depicting the roots, shoots, and yieldof corn plants symbiotic with ThLm1(S, right bars in each graph) ornonsymbiotic (NS, left bars in each graph) under drought stress.

FIG. 18 is a bar graph depicting root and shoot biomass in soybeanplants symbiotic with ThLm1 (S, left bar) or nonsymbiotic (NS, rightbar) under salt stress.

FIG. 19 is a bar graph depicting shoot and root biomass in cornsymbiotic with ThLm1 (S, left bar) or nonsymbiotic (NS, right bar) undersalt stress.

FIG. 20 is a bar graph depicting the growth of nonsymbiotic (NS) orThLm1 symbiotic (S) corn plants exposed to high temperatures. Growth ofsymbiotic (right bars in each pair) and nonsymbiotic (left bars in eachpair) corn plants was measured after two weeks of exposure to hightemperatures (45-50° C.).

FIG. 21 is a bar graph depicting the growth of nonsymbiotic (NS) orThLm1 symbiotic (S) tomato plants exposed to high temperatures. Growthof symbiotic (right bars in each pair) and nonsymbiotic (left bars ineach pair) corn plants was measured after two weeks of exposure to hightemperatures (45-50° C., right pair) or normal temperatures (25-30° C.,left pair).

FIG. 22 is a graph of the percent germination of corn seeds treated withThLm1 (S) or not treated with ThLm1 (NS) under high heat (50-55° C.).

FIG. 23 is a graph of the percent germination of soybean seeds treatedwith ThLm1 (S) or not treated with ThLm1 (NS) under high heat (50-55°C.).

FIG. 24 is a graph of the percent germination of corn seeds treated withThLm1 (S) or not treated with ThLm1 (NS) exposed to a cold temperature(5° C.) for 144 hours. Three corn varieties were tested (A, P & D).

FIG. 25 is a graph of the percent germination of soybean seeds treatedwith ThLm1 (S) or not treated with ThLm1 (NS) exposed to a coldtemperature (5° C. or 15° C.) for 72 hours.

FIG. 26 is a graph showing drought stress tolerance conferred to soybeanplants by 16 geographically diverse isolates of T. harzianum. NS(Nonsymbiotic) represents control plants without an endophyte and eachbar represents four replicates of 9 plants (n=36). Plants were exposedto 9 days of drought stress (complete lack of watering) and standarddeviations were less than 5%. No wilt=plants with full turgor nodifferent from plants grown without plant stress; Mild wilt=plants withfull turgor but mild leaf curling at tips; Permanent wilt point=severewilt that plants do not recover from when re-watered.

FIG. 27 is a representative image of drought tolerance conferred tosoybeans by T. harzianum isolates. Plants were exposed to 6 days ofdrought conditions and all NS plants wilted while E1 colonized plantsretained all turgor. The E1 colonized plants also continued to growduring the drought stress as seen by plant sizes.

FIG. 28 is a graph showing drought stress tolerance conferred to cornplants by 16 geographically diverse isolates of T. harzianum. NS(Nonsymbiotic) represents control plants without an endophyte and eachbar represents four replicates of 9 plants (n=36). Plants were exposedto 9 days of drought stress (complete lack of watering) and standarddeviations were less than 5%. No wilt=plants with full turgor nodifferent from plants grown without plant stress; Mild wilt=plants withfull turgor but mild leaf curling at tips; Permanent wilt point=severewilt that plants do not recover from when re-watered.

FIG. 29 is a graph showing salt stress tolerance conferred to soybeanplants by 16 geographically diverse isolates of T. harzianum. NS(Nonsymbiotic) represents control plants without an endophyte and eachbar represents four replicates of 9 plants (n=36). Plants were exposedto 14 days of salt stress (300 mM NaCl) with standard deviations<10%.All endophytes significantly increased plant root biomass compared tononsymbiotic control plants. All symbiotic plants were healthy while NSplants were chlorotic and wilting.

FIG. 30 is a graph showing salt stress tolerance conferred to soybeanplants by 16 geographically diverse isolates of T. harzianum. NS(Nonsymbiotic) represents control plants without an endophyte and eachbar represents four replicates of 9 plants (n=36). Plants were exposedto 14 days of salt stress (300 mM NaCl) with standard deviations<10%.All endophytes decreased water usage by more than 100% compared tononsymbiotic control plants. All symbiotic plants were healthy while NSplants were chlorotic and wilting.

FIG. 31 is a graph showing salt stress tolerance conferred to cornplants by 16 geographically diverse isolates of T. harzianum. NS(Nonsymbiotic) represents control plants without an endophyte and eachbar represents four replicates of 9 plants (n=36). Plants were exposedto 14 days of salt stress (300 mM NaCl) with standard deviations<10%.All endophytes decreased water usage by more than 300% compared tononsymbiotic control plants. All symbiotic plants were healthy while NSplants were chlorotic and wilting.

FIG. 32 is a graph showing salt stress tolerance conferred to cornplants by 16 geographically diverse isolates of T. harzianum. NS(Nonsymbiotic) represents control plants without an endophyte and eachbar represents four replicates of 9 plants (n=36). Plants were exposedto 14 days of salt stress (300 mM NaCl) with standard deviations<10%.All endophytes increased plant root biomass by 12-32% compared tononsymbiotic control plants. All symbiotic plants were healthy while NSplants were chlorotic and wilting.

FIG. 33 is a graph showing heat tolerance conferred to corn plants by 11geographically diverse isolates of T. harzianum. NS (Nonsymbiotic)represents control plants without an endophyte and each bar representsfour replicates of 9 plants (n=36). Plants were exposed to 10 days of45° C. root temperatures with standard deviations<10%. All endophytesincreased plant shoot height by 15-46% compared to nonsymbiotic controlplants. All symbiotic plants were healthy while NS plants were chloroticand wilting.

FIG. 34 is a graph showing heat tolerance conferred to corn plants by 11geographically diverse isolates of T. harzianum. NS (Nonsymbiotic)represents control plants without an endophyte and each bar representsfour replicates of 9 plants (n=36). Plants were exposed to 10 days of45° C. root temperatures with standard deviations<10%. All endophytesincreased plant leaf chlorophyll levels by 16-37% compared tononsymbiotic control plants. All symbiotic plants were healthy while NSplants were chlorotic and wilting.

FIG. 35 is a graph showing heat tolerance conferred to soybean plants by6 geographically diverse isolates of T. harzianum. NS (Nonsymbiotic)represents control plants without an endophyte and each bar representsfour replicates of 9 plants (n=36). Plants were exposed to 10 days of45° C. root temperatures with standard deviations<10%. All endophytesdecreased plant water use by 26-77% compared to nonsymbiotic controlplants. All symbiotic plants were healthy while NS plants were chloroticand wilting.

FIG. 36 is a graph showing heat tolerance conferred to soybean plants by6 geographically diverse isolates of T. harzianum. NS (Nonsymbiotic)represents control plants without an endophyte and each bar representsfour replicates of 9 plants (n=36). Plants were exposed to 10 days of45° C. root temperatures with standard deviations<10%. All endophytesincreased plant root biomass by 30-100% compared to nonsymbiotic controlplants. All symbiotic plants were healthy while NS plants were chloroticand wilting.

FIG. 37 is a graph showing cold tolerance conferred to soybean plants byan isolate (ThLm1) of T. harzianum. NS (Nonsymbiotic) represents controlplants without an endophyte and each bar represents three replicates of9 seeds (n=27, standard deviations<10%). Seeds were inoculated andplaced at either 5° C., 15° and 25° C. for 72 hours to assessgermination. The endophyte conferred cold tolerance and increased seedgermination at all temperatures compared to nonsymbiotic control plants.

FIG. 38 is a graph showing cold tolerance conferred to corn plants by anisolate (ThLm1) of T. harzianum. NS (Nonsymbiotic) represents controlplants without an endophyte and each bar represents three replicates of9 seeds (n=27, standard deviations<10%). Seeds of three corn varieties(A,P,D) were inoculated and placed at 15° C. 96 hours to assessgermination. The endophyte conferred cold tolerance and significantlyincreased seed germination compared to nonsymbiotic control plants.

FIGS. 39A-39C present a series of graphs showing the photosyntheticefficiency (Qmax, Y-axes) measured over time in nonsymbiotic (NS) andsymbiotic (TT=ThLM1) corn seedlings two weeks post germination. Plantswere grown without stress (FIG. 39A), or in the presence of droughtstress (FIG. 39B) or heat stress (FIG. 39C). In all cases, quantumefficiency was significantly higher in symbiotic plants.

FIG. 40 is a bar graph showing the average corn yield following droughtstress at one location in Michigan, USA. The site was subjected torandomized design with 25 plots (20 feet×20 feet each). Four cornvarieties were tested with 2 BioEnsure® (formulated ThLm1) treatments(L=low number of spores/seed & H=high number of spores/seed) and therewere 10 replicates/treatment with N=30/treatment). Seeds were inoculatedwith powder formulations of BioEnsure® and were planted within 2 hours.(All other field studies involved liquid formulations.) Treatment withBioEnsure® resulted in 30-85% increase in corn yield relative to plantsthat did not receive the treatment.

FIG. 41 is a bar graph showing the average corn yield with low stress atsites established by independent cooperators in several ecozones inMichigan and Indiana, USA. The sites were subjected to randomized designwith a total of 215 plots. Overall 84% of plots treated with BioEnsure®(formulated ThLm1) had an increase in corn yield with the average beinga 6.5% increase and a range of 3-20% increase. The increase inbushels/acre ranged from 5-38.

FIG. 42 is a bar graph showing the average rice yield with low stress inplots established in two soil types in Texas, USA. The sites weresubjected to randomized design with a total of 105 plots (20 feet×20feet each). Three rice varieties were tested with 2 BioEnsure®(formulated ThLm1) treatments, and there were 4 replicates/treatmentdrill seeded at 80 lb/acre. BioEnsure® induced yield increases rangedfrom 191-657 lbs/acre with a field average increase of 6% and a rangefrom 2.1-18.2%.

FIG. 43 is a bar graph showing the average barley yield in low stress.One barley variety was planted in 18 randomized plots (20 feet×20 feeteach). BioEnsure® (formulated ThLm1) treatment increased barley yield by7%.

FIG. 44 is a bar graph showing the grass yield in salt stress. A totalof 18 plots of grass were assessed in the presence of constant saltstress of 30-80 mM. The BioEnsure® (formulated ThLm1)-treated plants had86% more biomass than plants that did not receive the treatment(untreated).

FIG. 45 is a bar graph showing heat tolerance conferred to soybeanplants by 15 geographically diverse isolates of T. harzianum. NS(Nonsymbiotic) represents control plants without an endophyte and eachbar represents four replicates of 9 plants (n=36). Plant roots wereexposed to 10 days of 45° C. with standard deviations<10%. Allendophytes increased plant leaf chlorophyll levels by 16-37% compared tononsymbiotic control plants. All symbiotic plants were healthy while NSplants were chlorotic and wilting.

FIG. 46 is a bar graph showing heat tolerance conferred to soybeanplants by 15 geographically diverse isolates of T. harzianum. NS(Nonsymbiotic) represents control plants without an endophyte and eachbar represents four replicates of 9 plants (n=36). Plant roots wereexposed to 10 days of 45° C. with standard deviations<10%. Allendophytes increased plant biomass compared to nonsymbiotic controlplants. All symbiotic plants were healthy while NS plants were chloroticand wilting.

FIG. 47 is a bar graph showing nutrient tolerance conferred to soybeanplants by 16 geographically diverse isolates of T. harzianum. One-weekold soybean plants were given an initial watering with nitrogen,phosphorus, potassium (NPK), after which plants were exposed every 2days to no NPK stress (watered with full strength NPK) or high NPKstress (watered with ¼ strength NPK) for the duration of the experiments(approximately 90 days). Plants were then assessed for biomass (rootsand shoots) and data are shown for the high NPK stress plants. NS(nonsymbiotic) represents control plants without an endophyte.

FIG. 48 is a bar graph showing nutrient tolerance conferred to cornplants by 16 geographically diverse isolates of T. harzianum. One-weekold corn plants were given an initial watering with nitrogen,phosphorus, potassium (NPK), after which plants were exposed every 2days to no NPK stress (watered with full strength NPK) or high NPKstress (watered with ¼ strength NPK) for the duration of the experiments(approximately 90 days). Plants were then assessed for biomass (rootsand shoots) and data are shown for the high NPK stress plants. NS(nonsymbiotic) represents control plants without an endophyte.

FIG. 49 is a bar graph showing seed germination enhancement conferred tosoybean plants by 19 geographically diverse isolates of T. harzianum. UT(untreated) represents control seeds without an endophyte, and each barrepresents 20 seeds. Data represent the percent enhanced germinationabove untreated control seeds. All endophytes increased seed germination40-70× compared to untreated control seeds.

FIG. 50 is a bar graph showing cold tolerance conferred to corn plantsby 17 geographically diverse isolates of T. harzianum. Cold tolerance isexpressed as root radical length from germinated seeds. NS(Nonsymbiotic) represents control plants without an endophyte and eachbar represents 30 germlings exposed to 15° C. for 6 days with standarddeviations<10%. All endophytes increased germling growth by 10-32×compared to nonsymbiotic control plants. All symbiotic plants werehealthy while NS plants were stunted and desiccated.

FIG. 51 is a bar graph showing cold tolerance conferred to soybeanplants by 18 geographically diverse isolates of T. harzianum. Coldtolerance is expressed as seed germination at 15° C. NS (Nonsymbiotic)represents control seeds without an endophyte and each bar represents 18seeds exposed to 15° C. for 4 days with standard deviations<10%. Allendophytes increased seed germination by 85-240% compared tononsymbiotic control plants.

DETAILED DESCRIPTION OF THE INVENTION

In this century the foreseeable limitations on agricultural productivityinclude abiotic stresses such as drought, temperature extremes andsalinity. Currently, there are no stress tolerant crop plants orcommercial products capable of generating stress tolerant crops. Abioticstress tolerant crops have not been generated by breeding, mutationalselection or genetically modified (GM) approaches and remain a “holygrail” of agriculture.

Corn, soybean, wheat and rice are grown in great quantities around theworld and are considered to be among the most important crops forsustaining the human population. However, growers are faced with manychallenges in maintaining the high crop yields required for agriculturalsustainability. In recent years, global climate changes have contributedto an increased frequency of severe droughts in many agriculturalsettings. It is possible to mitigate the impacts of drought throughirrigation, however, this is a costly fix and water quality/availabilityis also decreasing.

There are several microbial products available that enhance growth,alter the nutrient status of plants or protect plants against microbialor insect pathogens (Table 1; Berg, 2009).

TABLE 1 Microbial products for use in agriculture Microbial Genus/GroupDescription Agriculture use category Ampelomyces Fungal Disease controlAzospirillum Bacterial Growth enhancement Bacillus Bacterial Diseasecontrol & Growth enhancement Beauveria Fungal Insect controlBradyrhizobium Bacterial Nutrient supplementation Candida Fungal Postharvest disease control Colletotrichum Fungal Weed control ConiothyriumFungal Disease control Delftia Bacterial Growth enhancement ErwiniaBacterial Disease control Metarhizium Fungal Insect control MycorrhizaeFungal Nutrient acquisition Paecilomyces Fungal Disease controlConiothyrium Fungal Disease control Phytophthora Fungal Weed controlPichia Fungal Post harvest disease control Pseudomonas Bacterial Diseasecontrol, Growth enhancement, Frost protection Rhizobacteria BacterialDisease control Rhizobium Bacterial Nitrogen supplementation SerratiaBacterial Disease control Streptomyces Bacterial Disease control, Growthenhancement Trichoderma Fungal Disease control

Trichoderma harzianum (T. harzianum) is a fungal species thatencompasses a wide variety of physiologically specialized strains, someof which are used as biopesticides against soil-borne plant pathogens orfor the industrial production of enzymes (Naseby et al., 2000). T.harzianum has been shown to induce metabolic changes in plants thatincrease their resistance to a wide variety of plant-pathogenicmicroorganisms and viruses (Harman et al., 2004). It has previously beenshown that T. harzianum strain T-22, can increase the growth of plantand root development under some conditions. T-22 can also solubilizeplant nutrients for plant uptake that would otherwise be unavailable toplants in certain soils (Altomare et al., 1999; Harman et al., 2004). Inaddition, another strain of T. harzianum TSTh20-1 (Patent DepositDesignation number PTA10317), isolated as an endophytic fungus from aplant that was found growing on oil-sand tailings, was found to promoteplant growth, particularly under sub-optimal or stressful conditionswith respect to water, organic carbon, nitrogen and mineral content,temperature, and contamination with polycyclic aromatic hydrocarbons ornaphthenic acids (PCT Publication No. WO2011/032281).

As described herein, a novel endophytic fungal symbiont Trichodermaharzianum strain, referred to herein as ThLm1, was isolated from a grass(Poacea) species growing on a beach in Washington state. This strain isnot a known pathogen of plants or animals.

When other plants, such as crop plants and native plants, wereinoculated with T. harzianum strain ThLm1, it was found that thisinoculation lead to several benefits including an increase in plant seedgermination rates, growth and yields; a decrease in water consumptionand NPK (nitrogen, phosphorus and potassium) supplementation; aconference of tolerance to abiotic stresses including drought,temperature, salinity; and an enhancement of general plant health. Itwas found that ThLm1 was capable of colonizing a broad range of plantsincluding both monocots and eudicots and as such, have cross plant/croputility. Additionally, it was found that T. harzianum strain ThLm1resided entirely within the plant vegetative tissues and did not growinto seeds or fruits of plants which had been inoculated. As a result,ThLm1 has cross-crop utility, can be used to provide consumer productsfree of the endophyte, and confers abiotic stress tolerance (salt,temperature, low NPK, drought), biomass and yield enhancement, andincreases overall plant health and robustness.

Other strains of T. harzianum are commercially used as biopesticides,plant growth enhancers and for the production of industrial enzymes. Nostrains have been reported to enhance seed germination or confersalinity or temperature stress tolerance, as has been found for ThLm1.Although other strains have been reported to decrease water consumption,ThLm1 produces superior results in agricultural crops and have beenshown to confer benefits under field conditions (see Examples).

Accordingly, aspects of the disclosure relate to isolated Trichodermaharzianum strain ThLm1, and compositions and methods of use thereof.

Additional aspects of the disclosure relate to methods of use ofTrichoderma harzianum as described here for strain ThLm1. Provided aremethods for increasing stress tolerance by inoculating a plant or aplant seed with an isolated T. harzianum fungus or a spore thereof; or acomposition comprising the isolated T. harzianum fungus or a sporethereof, thereby increasing stress tolerance of the inoculated plant. Insome embodiments, the stress is drought, elevated salt, reducednutrients and/or temperature stress and/or is not the presence ofpolycyclic aromatic hydrocarbons, napthenic acids, or high pH. Methodsfor increasing germination of seeds are also provided, in which plantseeds are inoculated with an isolated T. harzianum fungus or a sporethereof; or a composition comprising the isolated T. harzianum fungus ora spore thereof, thereby increasing germination of the inoculated seeds.Also provided are methods for reducing establishment of fungi other thanT. harzianum in a plant by inoculating a plant seed or seedling with anisolated T. harzianum fungus or a spore thereof; or a compositioncomprising the isolated T. harzianum fungus or a spore thereof, therebyreducing establishment of fungi other than T. harzianum in a plantgrowing from the inoculated seed or seedling. In some embodiments ofthese aspects, the T. harzianum fungus is not strain TSTh20-1 or strainT-22.

Definitions:

The term “abiotic stress” as used herein refers to the negative impactof physical and chemical factors on living organisms in a specificenvironment. Physical and chemical factors can include, but are notlimited to, water, organic nutrient levels, mineral nutrient levels,chemical contamination, chemical treatment (e.g., pesticides such asfungicides and insecticides), temperature, rainfall, pH, redox, oxygencontent, hydrocarbon residues, alkali, metals, salinity, atmosphericgases, light, soil composition.

The term “endophyte” as used herein refers to a class of fungalsymbionts that reside within host plant roots, stems and/or leaves.

The term “inoculating a plant” with a fungus, for example, as usedherein refers to applying or infecting a plant with a fungus or anyfungal developmental phase.

The term “plant” as used herein includes any member of the plant kingdomthat can be colonized by fungi. In one embodiment, the plant is anagricultural crop including, without limitation corn, soybean, rice,wheat, cotton, barley, sorghum, tomato, cucurbits, leafy greens, cotton,berries, woody plants and turf grasses. These plant species have beentested and T. harzianum strains such as ThLm1 has been shown to confergrowth benefits to them. In other embodiments, the plant is anornamental, such as Rosaceae, Liliaceae, Azalea, Rhododendron, Poaceaeor Chrysanthemum.

The term “progeny of ThLm1” as used herein refers to all cells derivingfrom ThLm1 cells.

The term “promoting plant growth” or “increased plant biomass andyields” as used herein means that the plant or parts thereof (such asroots and shoots, and seed/fruit yields) have increased in size, mass ornumber compared to control nonsymbiotic plants, or parts thereof, thathas not been inoculated with the fungus or as compared to apredetermined standard.

The term “spores of ThLm1” as used herein refers to asexual reproductivecells formed by ThLm1 fungi, or its sexual stage, Hypocrea.

The term “symbiosis” and/or “symbiotic relationship” as used hereinrefer to a beneficial interaction between two organisms including theinteraction most plants have with fungi such as mycorrhizae. Similarly,the term “symbiont” as used herein refers to an organism in a symbioticinteraction.

The term “water use efficiency” as used herein means the amount of wateror fluid consumed by plants over a defined period of time. It can alsobe defined by water or fluid use per gram of plant biomass or waterpotential.

Isolated Trichoderma Harzianum ThLm1

In one aspect, the disclosure relates to an isolated Trichodermaharzianum strain ThLm1 fungus, submitted to the U.S. Department ofAgriculture Culture Collection on Jul. 25, 2013 and deposited underPatent Deposit Designation number NRRL 50846 (date of deposit, Jul. 26,2013), or a progeny or spore thereof.

A progeny of ThLm1 includes any cell derived from a ThLm1 cell. A sporeof ThLm1 includes an asexual reproductive cell formed by ThLm1 fungi, orby its sexual stage, Hypocrea. As used herein, “isolated” refers to acell or fungus that has been removed from its natural symbiotic host.For example, an isolated Trichoderma harzianum strain ThLm1 may beremoved from a Poacea grass species. A fungus may be isolated usingmethods well-known in the art or described herein. The isolated fungi orcell may be maintained after isolation using methods known in the art,e.g., by culturing on 0.1× potato dextrose agar medium supplemented withampicillin, tetracycline, and streptomycin, and grown at 22° C. with a12 hr light regime. After 5-14 days of growth, conidia can be harvestedfrom plates by adding 10 ml of sterile water and gently scraping offspores with a sterile glass slide.

Mutants and variants of ThLm1 can provide the same (or better) benefitsthan the parent strain. Mutants can be generated using methods known tothose skilled in the art, such as by exposure to various chemicals,irradiation, physical conditions, molecular manipulation, viral basedDNA modifications and or plasmid based DNA modifications. Mutants canthen be selected by the methods described herein to identify mutantsthat provide the same or better benefits as the parent strain. Variantscould also be generated by exposing colonized plants to physical orchemical conditions and selecting for symbiotic benefits.

Compositions

Other aspects of the disclosure relate to compositions comprising aTrichoderma harzianum strain, such as ThLm1, and/or a progeny and/or aspore thereof.

In some embodiments, the composition may comprise a physiologicallyacceptable carrier, such a carrier that is not harmful to a seed and/orplant. Such carriers are known in the art (see, e.g., PCT PublicationNo. WO96/039844 and U.S. Pat. Nos. 5,586,411; 5,697,186; 5,484,464;5,906,929; 5,288,296; 4,875,921; 4,828,600; 5,951,978; 5,183,759;5,041,383; 6,077,505; 5,916,029; 5,360,606; 5,292,507; 5,229,114;4,421,544; and 4,367,609, each of which is incorporated herein byreference).

In addition, a variety of substances can be added during the treatmentof seeds including fungicides, insecticides, nematicides, bactericides,nutrients, biopesticides, other microbial inoculants, colorants,hydration matrices and polymers, all of which are well known in the art.Most of the substances commonly used in seed treatment do not interferewith the establishment of symbioses between Trichoderma harzianumstrains, such as ThLm1, and plant species.

In some embodiments, the composition is in a fluid form suitable forspray application or dip application. The composition may be diluted orconcentrated. In one embodiment, the composition is diluted with waterbefore inoculation. In another embodiment, the composition is in a pasteform. In still another embodiment, the composition is in a dry andpowdered form for dusting. The composition may be applied to any part ofthe plant including roots, leaves, stems or seeds. The composition ispreferably applied to dried seeds, such as in the form of a seedcoating.

One of skill in the art can readily determine the amount orconcentration of the composition that may be applied to the plant orplant seed to achieve a desired result, such as induction of stresstolerance and/or enhancement of growth of a plant. In one embodiment,from about 5 to about 100,000 viable spores of the T. harzianum strainssuch as ThLm1 can be used per seed, preferably between about 50 to 1000viable spores per seed.

Plants and Plant Seeds

Other aspects of the disclosure relate to plants and plant seeds thathave been inoculated with an isolated Trichoderma harzianum strain, suchas ThLm1, or a spore or progeny thereof as described herein. The term“plant” as used herein includes any member of the plant kingdom that canbe colonized by fungi. Determining whether a plant is colonized by aTrichoderma harzianum strain, such as ThLm1, can be performed usingstandard methods known to those skilled in the art, such as isolatingthe fungus from the plant and identifying it (by the methods known inthe art, such as described herein), direct DNA extraction from the plantfollowed by detection analysis (e.g., using PCR or probes). It may bedetermined that the plant is colonized by simple observation of itshealth and stress tolerance, or variation in physiological metricscompared to nonsymbiotic plants.

The plant or plant seed may be, e.g., a monocot or a eudicot. In someembodiments, the plant or plant seed is a crop plant or seed thereof,including, without limitation corn, soybean, rice, wheat, watermelon,tomato, cucurbits, peppers, leafy greens, barley, cotton, beans, pea,tubers and woody plants.

In some embodiments, the plant is a native plant. As used herein, anative plant is a plant that is indigenous or naturalized to a habitator climate zone. Examples of native plants include, but are not limitedto grasses, trees, flowering plants, shrubs, and herbs, such as spikebentgrass, annual hairgrass, California brome, slender hairgrass, bluewild rye, meadow barley, American dunegrass, American pokeweed, andArabidopsis. A person of skill in the art can readily identify nativeplants for a particular habitat or climate zone, e.g., using knownclassification systems and databases (see, e.g., the Native PlantDatabase at wildflower.org; the Native Plant Network atnativeplantnework.org; the Plants database through the United StatesDepartment of Agriculture; Kenrick, Paul & Crane, Peter R. (1997). TheOrigin and Early Diversification of Land Plants: A Cladistic Study.Washington, D.C.: Smithsonian Institution Press. ISBN 1-56098-730-8;Raven, Peter H., Evert, Ray F., & Eichhorn, Susan E. (2005). Biology ofPlants (7th ed.). New York: W. H. Freeman and Company; and Prance G. T.(2001). “Discovering the Plant World”. Taxon (International Associationfor Plant Taxonomy) 50 (2, Golden Jubilee Part 4): 345-359.).

In some embodiments, the plant is a non-native plant. As used herein, anon-native plant is a plant that is not indigenous to a habitat orclimate zone and may be naturalized or invasive. Examples of non-nativeplants include, but are not limited to grasses, trees, flowering plants,shrubs, and herbs, succulents, etc. A person of skill in the art canreadily identify native plants for a particular habitat or climate zone,e.g., using known classification systems and databases (see, e.g., thePlants database through the United States Department of Agriculture).

A plant or plant seed may be inoculated using any method known in theart or described herein. A plant may be tested for inoculation with a T.harzianum strain, such as ThLm1, or a spore or progeny thereof, e.g., byisolating the fungus from the plant using standard procedures andidentifying the fungus by the methods described herein, by direct DNAextraction from the plant followed by detection analysis, or byobservation of plant health and stress tolerance, or variation inphysiological metrics compared to nonsymbiotic plants.

Methods

Yet other aspects of the disclosure relate to method for promotingstress tolerance and/or enhancing plant growth or seed germination. Insome embodiments, the method comprises inoculating a plant or a plantseed with an isolated Trichoderma harzianum strain, such as ThLm1, or aspore or progeny thereof.

The term “stress” as used herein refers to the negative impact ofphysical, organic, and chemical factors on living organisms, such asplants, in a specific environment, including impact on seed germination,plant growth or development, and/or plant (e.g., fruit) yield. Physicaland chemical factors can include, but are not limited to, water(including soil moisture and rainfall), nutrient levels (includingorganic and mineral nutrient levels, and especially low nutrientlevels), temperature (including high or low temperatures that areoutside of the range of temperatures tolerated by a particular plant oroptimal for a particular plant), pH (including high or low pH that areoutside of the range of pH tolerated by a particular plant or optimalfor a particular plant), Redox, oxygen content, salinity, atmosphericgases, light, and soil composition. Organic factors can include fungi,bacteria, viruses, nematodes or insects which may be pathogenic, orother animals which may be herbivores. As used herein “stress tolerance”refers to resistance of a living organism, such as a plant, to a stresscondition. The resistance may be, e.g., an ability to grow or persist instress conditions that would otherwise result in death of the plant orgrowth reduction and/or yield reduction in the plant. In someembodiments, the stress tolerance is drought tolerance, salt tolerance,reduced nutrient tolerance, fungal tolerance, and/or temperaturetolerance. In some embodiments, plants that are inoculated with T.harzianum strains, such as ThLm1, are more stress tolerance relative toplants that are not inoculated with said strains.

An increase or promotion of stress tolerance may be determined bycomparing a level of stress tolerance in a plant inoculated with T.harzianum strains, such as ThLm1 or a spore or progeny thereof, with alevel of stress tolerance in a control plant that has not beeninoculated. For example, the growth rate, germination rate, yield, mass,physiological metrics and other such factors may be measured in aninoculated plant under stress conditions, and compared to the samefactors in a control non-inoculated plant under similar stressconditions. An increase of one or more such factors in the inoculatedplant would indicate an increase or promotion of stress tolerance. Anincrease or promotion of stress tolerance may also be determined bycomparing a level of stress tolerance in a plant inoculated with T.harzianum strains, such as ThLm1 or a spore or progeny thereof, with apredetermined standard. The predetermined standard may be, e.g., anaverage growth rate, germination rate, photosynthetic efficiency, yieldor mass of a population of plants, such as a population ofnon-inoculated plants. In some embodiments, promoting stress tolerancecomprises an increase in stress tolerance of at least 5%, 10%, 15%, 20%,25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, ormore compared to a control or predetermined standard.

The term “enhancing growth” as used herein refers to a plant or partsthereof (such as roots and shoots) that have an increase in size, extentof root development (e.g., how extensive a root system is), germinationrate of seeds, chlorophyll content/level, yield (e.g., number of seeds,or fruit production) or mass compared to control plants, or partsthereof, that has not been inoculated with the fungus or as compared toa predetermined standard. The predetermined standard may be, e.g., anaverage size, extent of root development, germination rate, chlorophyllcontent/level, photosynthetic efficiency, yield or mass of a plant orplant part of a population of plants, such as a population ofnon-inoculated plants. Determining an enhancement in plant growth can beassessed in a number of ways. For example, the size, extent of rootdevelopment, germination rate, chlorophyll content/level, photosyntheticefficiency, yield or mass of the entire plant or a part thereof (such asseeds, fruit, shoots and roots) can be measured. In some embodiments,enhancing growth comprises increasing size, extent of root development,germination rate, chlorophyll content/level, photosynthetic efficiency,yield or mass of a plant or parts thereof by at least 5%, 10%, 15%, 20%,25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, ormore than a control or predetermined standard.

As used herein, “inoculating a plant or plant seed” with a fungus, forexample, refers to applying or colonizing a plant or plant seed with afungus or a fungal cell from any fungal developmental phase, such as aspore. In some embodiments, inoculating the plant comprises colonizing aroot and/or stem of the plant with a Trichoderma harzianum strain, suchas ThLm1.

In some embodiments, a method provided herein further comprises growingthe plant or plant seed. The plant or plant seed may be grown in one ormore stress conditions, such as drought conditions, high saltconditions, reduced nutrient conditions, low light conditions, parasiticfungal conditions, and/or high temperature conditions.

Thus, in some embodiments, a method provided herein comprises growingthe plant or plant seed in soil characterized by high salinity, lowmoisture, and/or low nutrient content. Soil characterized by highsalinity includes soil that has greater than 35 mM amount of a salt,such as Na, Mg, Ca or K chlorides, sulfates or carbonates, present inthe soil, such as greater than: 35 mM, 40 mM, 45 mM, 50 mM, 60 mM, 70mM, 80 mM, 90 mM, 100 mM, 120 mM, 140 mM, 160 mM, 180 mM, 200 mM, 250mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM or more amount of a salt.Soil characterized by low moisture includes soil that has between 0-0.18in³ water/in³ soil depending on soil type, such as less than: 0.18,0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06,0.05, 0.04, 0.03, 0.02 or 0.01 in³ water/in³ soil. Soil characterized bylow nutrient content includes soil that has fewer than 80 lbs/acre ofnutrient (comprising 20% each of nitrogen, phosphorus, and potassium(NPK), and associated micronutrients), such as fewer than: 75, 70, 65,60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10 or 5 lbs/acre of nutrient.

In some embodiments, a method provided herein comprises growing theplant or plant seed at an average temperature at or above 36 degreesCelsius, such as 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50 or higher. In some embodiments, the method comprises germinating theplant seed at an average temperature at or above 36 degrees Celsius,such as 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 orhigher.

In some embodiments, a method provided herein comprises growing theplant or plant seed at an average temperature at or below 15 degreesCelsius, such as 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or lower. In someembodiments, the method comprises germinating the plant seed at anaverage temperature at or below 15 degrees Celsius, such as 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5 or lower.

In some embodiments of any of the methods provided herein, the plant orplant seed is a crop plant or crop plant seed. In some embodiments, thecrop plant or crop plant seed is watermelon, tomato, corn, wheat,soybean, cucurbits, peppers, leafy greens, barley, cotton, beans, pea,tubers, woody plants, berries or rice, or a seed thereof. In otherembodiments, the plant or plant seed is an ornamental, such as Rosaceae,Liliaceae, Azalea, Rhododendron, Poaceae or Chrysanthemum.

Unless defined otherwise, all technical and scientific terms used hereinare intended to have the same meaning as is commonly understood by oneof ordinary skill in the relevant art.

As used herein, the singular forms “a,” “an”, and “the” include theplural reference unless the context clearly dictates otherwise.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present invention toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever. All publicationscited herein are incorporated by reference for the purposes or subjectmatter referenced herein.

EXAMPLES Example 1 Isolation and Identification of ThLm1 as a BeneficialSymbiont Isolation and Inoculation of ThLm1

Trichoderma harzianum strain ThLm1 was isolated from a grass (Poacea)species growing on a beach in Washington state. The habitat-stresses onthese plants were identified as drought, low soil nutrients, andelevated salinity. Leymus mollis (dunegrass) plants were collected fromseveral coastal beach habitats in the San Juan Island Archipelago, Wash.Plants were washed until soil debris was removed, placed into plasticzip-loc baggies and surface sterilized as previously described (Redmanet al., 2001, 2002a). Using aseptic technique, plants were cut intosections representing the roots, rhizomes and stems, plated on fungalgrowth media [0.1× PDA (potato dextrose agar)] and incubated at roomtemperature for 5-7 days under cool fluorescent lights to allow for theemergence of fungi. Upon emergence, 30 representative isolates of thedominant fungal endophyte represented (×95%) were sub-cultured and, ofthese, single spore isolation of 10 representative isolates wasperformed as previously described (Redman et al., 1999). All 10 of therepresentative isolates were placed under sterile water supplementedwith 50-100 mg/ml of ampicillin in sterile 1.5 ml screw-cap tubes andplaced at 4° C. for long-term storage.

Colonization of Seeds and Plants with ThLm1

Several test plants were inoculated with ThLm1. Corn (Zea mays) andsoybean (Glycine max) seeds were surface-sterilized in a 0.5-1.0% (v/v)sodium hypochlorite solution for 15-20 min with moderate agitation andrinsed with 10-20 volumes of sterile distilled water. These plantspecies represent two major plant lineages monocots and eudicots.However, ThLm1 also establishes symbioses with many other plantsincluding vegetables (e.g., tomato, cucurbits, peppers, pea, beans,turf, leafy greens etc.), staple crops (e.g., wheat, rice, barley,tubers), grasses and woody plants, which are treated as described forcorn and soybean to colonize with ThLm1. Plants can establish symbioseswith ThLm1 by treating (sprayed, soaked, mixed or watered) seeds,germlings, seedlings or older stage plants directly with either liquidor powdered formulations of spores (10-10,000 spores/seed). Spores canbe applied to any part of the plant with best results obtained byinoculating lower stem and roots.

ThLm1 was found to establish symbiosis with many plant speciesinoculated with ThLm1 including crop plants and native plants.Inoculation with ThLm1 was found to confer stress tolerance. A summaryof the plants that were inoculated with ThLm1 and had improved stresstolerance are shown in Table 2.

TABLE 2 Plants inoculated with ThLm1 Common Name Colonization Effect onPlants Interaction Crop Species Citrullus lanatus subsp. vulgariswatermelon roots, stem stress tolerance mutualism Solanum lycopersicumtomato roots, stem stress tolerance mutualism Zea mays subsp. mays cornroots, stem stress tolerance mutualism Triticum aestivum wheat roots,stem stress tolerance mutualism Glycine max soybean roots, stem stresstolerance mutualism Oryza sativa rice roots, stem stress tolerancemutualism Native Species Agrastis exarata spike bentgrass roots, stem,leaf stress tolerance mutualism Deschampsia danthonioides annualhairgrass roots, stem, leaf stress tolerance mutualism Bromus carinatuscalifornia brome roots, stem, leaf stress tolerance mutualismDeschampsia elongata slender hairgrass roots, stem, leaf stresstolerance mutualism Elymus glaucus Buckley blue wild rye roots, stem,leaf stress tolerance mutualism Hordeum brachyantherum Nevski meadowbarley roots, stem, leaf stress tolerance mutualism Leymus mollisAmerican dunegrass roots, stem, leaf stress tolerance mutualismPhytolacca americana American pokeweed roots, stem, leaf stresstolerance mutualism Arabidopsis thaliana Arabidopsis roots, stem, leafstress tolerance mutualism

ThLm1 Increased Plant Growth and Health in the Absence of Stress

Corn plants were inoculated or not inoculated with ThLm1 and were grownin the absence of stress conditions in a greenhouse for one month. Thecorn plants were then separated into root and shoot sections for wetweight measurements. Statistical analysis of the weight measurementsshowed that plants inoculated with ThLm1 (symbiotic with ThLm1) weresignificantly larger than non-inoculated plants (ANOVA, P<0.05, FIG. 1).

Corn growth and yield were also measured in corn plants inoculated ornot inoculated with ThLm1. Mature greenhouse plants were further grownin the absence of stress to produce corn ears (yields) for wet weightmeasurements. Statistical analysis showed that plants inoculated withThLm1 (symbiotic with ThLm1) produced significantly more yield thannon-inoculated plants (N=48; P>0.05, FIG. 2).

Corn growth and yield enhancement in ThLm1 inoculated corn grown in theabsence of stress was further measured. Mature greenhouse plants grownin the absence of stress were separated into whole plant weight(biomass) or corn ears (yields) for wet weight measurements. Statisticalanalysis showed that plants inoculated with ThLm1 (symbiotic with ThLm1)were significantly larger than non-inoculated plants (N=48; P>0.05, FIG.3).

Seed germination enhancement in ThLm1 inoculated plants was alsomeasured. Germination of corn seeds was measured in the presence andabsence of stress in plant growth incubators and greenhouses.Statistical analysis showed that seeds inoculated with ThLm1 (symbioticwith ThLm1) had significantly higher rates of germination thannon-inoculated (NS) seeds (N=100; SD<5; P<0.05, FIGS. 4, 22-25).

Chlorophyll content was also measured in ThLm1 inoculated plants. Maturegreenhouse corn plants grown in the absence of stress were measured for% chlorophyll content. Statistical analysis showed that ThLm1 inoculatedplants (symbiotic with ThLm1) had higher chlorophyll content thannon-inoculated plants (N=9; P>0.05, FIGS. 5, 13, 14). As a generalobservation, ThLm1 inoculated plants looked greener and more robust thannon-inoculated plants.

These data show that ThLm1 inoculation of plants generally increasedplant health, including plant growth, yield, germination rates, andchlorophyll content in the absence of stress.

ThLm1 Increased Plant Growth and Health in the Presence of Stress

Stress conditions were also tested to determine if plants inoculatedwith ThLm1 were healthier than plants not inoculated with ThLm1. Rootand shoot weights were measured in corn plants exposed to drought stressthat were previously inoculated or not inoculated with ThLm1. Sixty-dayold corn plants were exposed to no stress (watered fully every 2 days),moderate drought stress (watered every 4 days) or high drought stress(watered every 7 days). Plants were then assessed for biomass from highdrought exposed plants (roots and shoots). Plants were separated intoroot and shoot sections for wet weight measurements. Statisticalanalysis showed that plants inoculated with ThLm1 (symbiotic with ThLm1)were significantly larger than non-inoculated plants (N=36; P>0.05, FIG.6A).

Root and shoot measurements were also taken in corn plants exposed tolow nutrient stress that were previously inoculated or not inoculatedwith ThLm1. One-week old corn plants were given an initial watering withnutrients (nitrogen, phosphorus & potassium based plant fertilizer,NPK), after which plants were exposed every 2 days to no nutrient stress(watered with full strength NPK) or low nutrient stress (watered with ¼strength NPK) for the duration of the experiments (approximately 90days). Plants were then assessed for biomass (roots and shoots). Plantswere separated into root and shoot sections for wet weight measurements.Statistical analysis showed that plants inoculated with ThLm1 (symbioticwith ThLm1) were significantly larger than non-inoculated plants (N=36;P>0.05, FIG. 6B).

Weight measurements were taken in corn plants under salt stress thatwere previously inoculated or not inoculated with ThLm1. Sixty-day oldplants were exposed every 2 days to no salt stress (water with standardlevels of NPK) or salt stress (200 mM NaCl in standard NPK solution) forthirty days. Plant wet weights were measured. Statistical analysisshowed that plants inoculated with ThLm1 (symbiotic with ThLm1) weresignificantly larger than non-inoculated plants (N=36; P≥0.05, FIG. 7).

Cold stress tolerance was measured in rice seedlings that werepreviously inoculated or not inoculated with ThLm1. The rice seedlingswere exposed to cold-water stress. After 2 weeks, rice seedlings wereassessed for root and shoot biomass (wet g). Rice inoculated with ThLm1had statistically larger roots and shoots (N=10; P<0.05, FIG. 8).

A field evaluation under drought stress was also conducted. Plants weregrown under dryland cultivation in Michigan, USA, and exposed to theworst drought in 50 years. Yield data was obtained for four replicateplots containing 120 corn plants each. Plants inoculated with ThLm1(symbiotic with ThLm1) produced an average of 85% more yield thannon-inoculated plants, FIG. 9.

These data show that inoculation with ThLm1 improved plant health, suchas growth and yield, under stress conditions including drought, lownutrient, temperature, and salt stress.

Summary

Taken together, the data show that inoculation with ThLm1 increased seedgermination rates and seedling growth, conferred tolerance to variousstresses (salt, temperature, drought, nutrients), enhanced generalrobustness and health (such as an increase in % chlorophyll), andincreased yields. In addition, it was found (data not shown) that ThLm1could competitively out compete plant colonization by other microbes andin so doing, thwart the ingress of pathogenic organisms (therebydecreasing incidence of disease) in a wide range or agricultural plantssuch as soybean, rice and corn. A list of benefits that ThLm1 providedto plants upon inoculation is summarized in Table 3.

TABLE 3 Th-Lm1 Inoculated Plant benefits Inoculated PlantCharacteristics Improved seed germination Drought tolerance Salttolerance Temperature tolerance Decreased nutrient requirementsIncreased photosynthetic efficiency Increased chlorophyll content Fieldbenefits conferred in corn, rice, wheat, barley, soybean Preventcolonization by other fungi Decreased water consumption Enhanced plantgrowth

Example 2 Isolation and Inoculation of Plants with ThLm1 Isolation ofThLm1

To isolate ThLm1, plants were washed until soil debris was removed,placed into plastic Zip-loc baggies and surface sterilized as previouslydescribed (Redman et al., 2001, 2002a). Using aseptic technique, plantswere cut into sections representing the roots, rhizomes and stems,plated on fungal growth media [0.1× PDA (potato dextrose agar, Difco)]and incubated at room temperature for 5-7 days under cool fluorescentlights to allow for the emergence of fungi. Upon emergence, 30representative isolates of the dominant fungal endophyte representedwere sub-cultured and, of these, single spore isolation of 10representative isolates was performed as previously described (Redman etal., 1999). All 10 of the representative isolates were placed understerile water supplemented with 50-100 mg/ml of ampicillin in sterile1.5 ml screw-cap tubes and placed at 4° C. for long-term storage.

Culturing ThLm1:

ThLm1 was cultured on 0.1× potato dextrose agar (PDA) medium (Difco).The medium was supplemented with 50-100 ug/ml of ampicillin,tetracycline, and streptomycin, and fungal cultures grown at 22° C. witha 12 hr light regime. After 5-14 days of growth, conidia were harvestedfrom plates by adding 10 ml of sterile water and gently scraping offspores with a sterile glass slide. The final volume of spores wasadjusted to 100 ml with sterile water, filtered through four layers ofsterile cotton cheesecloth gauze and spore concentration adjusted to10⁴-10⁵ spores/ml.

Identification of ThLm1:

Fungi were identified using conidiophore and conidial morphology (Arx,1981; Barnett and Hunter, 1998; Leslie and Summerell, 2005). Onceisolates from L. mollis were identified as the same fungal speciesmicroscopically, three of the isolates were randomly selected formolecular species identification. Species designations were based onsequence analysis of the variable ITS1 and ITS2 sequences of rDNA [ITS4(5′-tcctccgcttattgatatgc-3′; SEQ ID NO:1)/ITS5(5′-ggaagtaaaagtcgtaacaagg-3′; SEQ ID NO:2) primers] and translationelongation factor [EF1T (5′-atgggtaaggaggacaagac-3′; SEQ ID NO:3)/EF2T(5′-ggaagtaccagtgatcatgtt-3′; SEQ ID NO:4) and EF11(5′-gtggggcatttaccccgcc-3′; SEQ ID NO:5)/EF22(5′-aggaacccttaccgagctc-3′; SEQ ID NO:6) primers (O'Donnell et al.,2000; White et al., 1990)]. DNA was extracted from mycelia and PCRamplified as previously described (Rodriguez, 1993; Rodriguez and Yoder,1991). PCR products were sequenced and BLAST searched against theGenBank database. Morphological and GenBank analysis identified thethree isolates/species as the same species (Trichoderma harzianum) andall isolates tested for plant benefits.

Benefits of Trichoderma Harzianum Strain ThLm1

A series of studies were performed to assess the benefits of Trichodermaharzianum strain ThLm1 on a monocot (corn) and eudicot (soy and/ortomato) to demonstrate that the fungus establishes a beneficialsymbiosis with plants of both lineages.

ThLm1 Increased Plant Growth and Health in the Absence of Stress

Firstly, experiments were performed in the without stress conditions todetermine the general plant health, such as plant growth anddevelopment, of ThLm1 inoculated plants in the absence of stress.

Corn seedlings inoculated or not inoculated with ThLm1 were assessed forshoot and root biomass after 12 weeks of growth. Growth enhancement wasobserved in plants inoculated with ThLm1 (N=48; SD<6.8; P<0.050, FIG.10). Additionally, the corn inoculated with ThLm1 was found to produce amore extensive root system with more lateral roots (FIG. 11).

Soybeans seedlings inoculated or not inoculated with ThLm1 were alsoassessed for shoot and root biomass after 13 days of growth. Growthenhancement of soybean seedlings was observed in plants inoculated withThLm1 (N=10; SD<10%; P<0.050, FIG. 12).

Corn plants inoculated or not inoculated with ThLm1 were assessed forchlorophyll content. SPAD measurements were taken which indicatedrelative % chlorophyll. Corn plants inoculated with ThLm1 were found tohave a higher chlorophyll content than non-inoculated plants (N=60,P<0.05, FIG. 13). Generally, corn plants inoculated with ThLm1 weredarker green than non-inoculated plants.

Soybean plants inoculated or not inoculated with ThLm1 were alsoassessed for chlorophyll content. Soybean plants inoculated with ThLm1had significantly more chlorophyll than non-inoculated plants (N=10,SD<5%, P<0.05, FIG. 14).

Corn seeds inoculated or not inoculated with ThLm1 were assessed forgermination. Corn seeds inoculated with ThLm1 had a higher % ofgermination (N=100; SD<5; P<0.05, FIG. 4).

Corn plants inoculated or not inoculated with ThLm1 were assessed forfruit yield. For corn yield assessment, 6 corn plants were pooled todetermine yield weights of ears. ThLm1 inoculated plants produced morefruit than non-inoculated plants (N=6, P<0.05, FIG. 15).

Together, these data show that in the absence of stress ThLm1inoculation conferred growth enhancement, more extensive root systems,increased chlorophyll levels, higher germination rates, and higher fruityields.

ThLm1 Increased Plant Growth and Health in the Presence of Stress

Next, experiments were performed under stress conditions to determinethe general plant health of ThLm1 inoculated plants in the presence ofstress.

Corn plants inoculated or not inoculated with ThLm1 were assessed forroot mass, shoot mass, seedling weight, and yield under low nutrientstress. One-week old corn plants were given an initial watering withnitrogen, phosphorus, potassium (NPK), after which plants were exposedevery 2 days to no NPK stress (watered with full strength NPK), low NPKstress (watered with ½ strength NPK), or high NPK stress (watered with ¼strength NPK) for the duration of the experiments (approximately 90days). Plants were then assessed for yields and biomass (roots andshoots). ThLm1 inoculated plants had higher root mass, shoot mass,seedling weight, and yields than non-inoculated plants under lownutrient stress (N=12-60; SD<10%, P<0.05, FIG. 16).

Corn plants inoculated or not inoculated with ThLm1 were assessed forroot mass, shoot mass, and yield under drought stress. Sixty-day oldcorn plants were exposed to no stress (watered fully every 2 days) orhigh drought stress (watered every 7 days). Plants were then assessedfor yields and biomass (roots and shoots). ThLm1 symbiotic plants hadhigher root mass, shoot mass, and yields than NS plants under droughtstress (N=12-60; SD<10%, P<0.05, FIG. 17).

Soybean plants inoculated or not inoculated with ThLm1 were assessed forroot and shoot biomass under salt stress. Eight inoculated and eightnon-inoculated seedlings were exposed to salt (300 mM) for two weeks.Inoculated plants produced significantly more shoot and root biomass(FIG. 18). Non-inoculated plants leaves were wilted at the end of thestudy while inoculated plants remained healthy.

Corn plants inoculated or not inoculated with ThLm1 were assessed forroot and shoot biomass under salt stress. Eight inoculated and eightnon-inoculated seedlings were exposed to salt (300 mM) for two weeks.Inoculated plants produced significantly more shoot and root biomass(FIG. 19). Non-inoculated plants leaves were wilted at the end of thestudy while inoculated plants remained healthy.

Corn plants inoculated or not inoculated with ThLm1 were assessed forgrowth under high temperature stress. Growth was measured after twoweeks of exposure to high temperatures (45-50° C.). ThLm1 inoculatedplants grew more than non-inoculated plants under heat stress (FIG. 20).Non-inoculated plants were wilted while the inoculated plants remainedhealthy.

Tomato plants inoculated or not inoculated with ThLm1 were assessed forgrowth under high temperature stress. Growth was measured after twoweeks of exposure to high temperatures (45-50° C.). ThLm1 inoculatedplants grew more than non-inoculated plants under heat stress (FIG. 21).Non-inoculated plants were wilted while the inoculated plants remainedhealthy.

Corn seeds treated or not treated with ThLm1 were assessed forgermination under high temperature stress (50-55° C.). Seeds (N=15)treated with ThLm1 germinated significantly better than untreatedcontrol seeds (FIG. 22).

Soybean seeds treated or not treated with ThLm1 were assessed forgermination under high temperature stress (50-55° C.). Seeds (N=15)treated with ThLm1 germinated significantly better than untreatedcontrol seeds (FIG. 23).

Corn seeds treated or not treated with ThLm1 were assessed forgermination under low temperature stress. Corn seeds were exposed to acold temperature (5° C.) for 144 hours. Seeds (N=30) treated with ThLm1germinated significantly better than untreated control seeds (FIG. 24).

Soybean seeds treated or not treated with ThLm1 were assessed forgermination under low temperature stress. Soybean seeds were exposed toa cold temperature (5° C. or 15° C.) for 72 hours. Seeds (N=30) treatedwith ThLm1 germinated significantly better than untreated control seeds(FIG. 25).

Together, these data suggest that inoculation with ThLm1 improved planthealth and growth, including root and shoot mass, yield, andgermination, under stress conditions such as drought, salt, hightemperature, and low temperature.

ThLm1 Increases the Photosynthetic Efficiency in the Presence andAbsence of Stress

The photosynthetic efficiency (Qmax) is the fraction of light energyconverted into chemical energy during photosynthesis. Several studieswith ThLm1 in both the presence or absence of stress revealed thattreatment of plants with ThLm1 resulted in an increase in Qmax in bothmonocots and eudicots, as shown for example in FIG. 39A-39C for corn.

ThLm1 Increases Crop Yields in the Presence and Absence of Stress

Two years of field tests were performed in 8 states with BioEnsure®(formulated ThLm1) inoculated and non-inoculated seeds of corn, rice,grains, and grass (FIGS. 40-44). Field testing was performed underconditions of high drought and salt stress, or very low stress levels(normal growing season). BioEnsure® increased crop yields under allconditions although greater yield increases were observed under highlevels of stress. Yield increases occurred irrespective of soil type andclimate zone.

Summary

Taken together, the data show that inoculation with ThLm1 increased seedgermination rates and plant growth, conferred tolerance to variousstresses (salt, temperature, drought, nutrients), enhanced generalrobustness and health (such as an increase in % chlorophyll), andincreased yields.

Example 3 Effects of T. Harzianum Isolates on Increasing StressTolerance, Increasing Germination and Excluding Other Fungi

Strains of T. harzianum isolated from a diversity of geographiclocations (the origin of 16 of the strains are noted in Table 4) wereused to assess benefits of T. harzianum strains in addition to ThLm1,using methods as described in the examples above.

TABLE 4 Geographic Diversity of T. harzianum strains used Strain OriginE1 USA, WA E2 USA, NY E3 USA, OH E4 USA, FL E5 USA, TX E6 USA, GA E7USA, AL E8 Columbia E9 India E10 France E11 Belgium E12 Tokyo E13 UnitedKingdom E14 French Guiana E15 Spain E16 Canada

The results are depicted in FIGS. 26-38, 45-48, 50 and 51 for droughtstress tolerance, salt stress tolerance, temperature tolerance (heat andcold tolerance), and low nutrient tolerance conferred to plants bygeographically diverse isolates of T. harzianum. Not all of thegeographically diverse isolates of T. harzianum were used in eachexperiment or condition, but the results clearly support the use of T.harzianum generally for increasing tolerance to a variety of stresses.

Soybean plants inoculated or not inoculated with geographically diverseisolates of T. harzianum were assessed for growth under high temperaturestress. Growth was measured after two weeks of exposure to hightemperatures (45-50° C.). Plants inoculated with geographically diverseisolates of T. harzianum had increased leaf chlorophyll levels andhigher plant biomass more than non-inoculated plants under heat stress(FIGS. 45 and 46).

Soybean or corn plants inoculated or not inoculated with geographicallydiverse isolates of T. harzianum were assessed for seedling weight underlow nutrient stress. One-week old soybean or corn plants were given aninitial watering with nitrogen, phosphorus, potassium (NPK), after whichplants were exposed every 2 days to no NPK stress (watered with fullstrength NPK), low NPK stress (watered with ½ strength NPK), or high NPKstress (watered with ¼ strength NPK) for the duration of the experiments(approximately 90 days). Plants were then assessed for seedling weight.Plants inoculated with geographically diverse isolates of T. harzianumhad higher seedling weight than non-inoculated plants under low nutrientstress (FIG. 47, soybean; FIG. 48, corn).

Further, cold stress tolerance was measured in corn seedlings that werepreviously inoculated or not inoculated with geographically diverseisolates of T. harzianum. The corn seedlings were exposed to cold stressthen assessed for germling radical length. Corn inoculated with T.harzianum had statistically longer germlings (FIG. 50).

Cold stress tolerance also was measured in soybean seedlings that werepreviously inoculated or not inoculated with geographically diverseisolates of T. harzianum. The soybean seedlings were exposed to coldstress then assessed for germling radical length. Soybean seedlingsinoculated with T. harzianum had statistically longer germlings (FIG.51).

In other experiments, geographically diverse isolates of T. harzianumwere tested for their effects on germination of seeds, using themethodology described herein, as shown in FIG. 49. Soybean seedsinoculated or not inoculated with geographically diverse isolates of T.harzianum were assessed for germination. Soybean seeds inoculated withgeographically diverse isolates of T. harzianum had a significantlyhigher % of germination compared to soybean seeds that were untreated(FIG. 49). The results support the use of T. harzianum generally forincreasing germination of seeds.

In other experiments, the 16 geographically diverse isolates of T.harzianum are tested for their effects on establishment of fungi otherthan Trichoderma harzianum in a plant. Seeds or seedlings are inoculatedwith isolated T. harzianum isolates or spores thereof and the seeds orseedlings are grown. The growth of fungi other than the inoculated T.harzianum isolates is evaluated. The results support the use of T.harzianum generally for reducing establishment of fungi other thanTrichoderma harzianum in a plant growing from the inoculated seed orseedling.

REFERENCES

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Appl Microbiol Biotechnol 84:11-18.-   Clay K, Holah J (1999) Fungal endophyte symbiosis and plant    diversity in successional fields. Science 285: 1742-1745.-   Harman G E, Howell C R, Viterbo A, Chet I, Lorito M (2004)    Trichoderma species—opportunistic, avirulent plant symbionts. Nat    Rev Microbiol. 2: 43-56.-   Leslie J F, Summerell B A (2005). The Fusarium Laboratory Manual.    Blackwell Publishing: Ames, 400pp.-   Naseby D C, Pascual J A, Lynch J M (2000) Effect of biocontrol    strains of Trichoderma on plant growth, Pythium ultimum polulations,    soil microbial communities and soil enzyme activities. J Appl    Microbiol 88: 161-169.-   O'Donnell K, Kistler H C, Tacke B K, Casper H C (2000). Gene    genealogies reveal global phylogeographic structure and reproductive    isolation among lineages of Fusarium graminearum, the fungus causing    wheat scab. Proceedings of the National Academy of Sciences 97:    7905-7910.-   Petrini O (1996) Ecological and physiological aspects of    host-specificity in endophytic fungi. In: Redlin S C, Carris L M,    editors. Endopytic Fungi in Grasses and Woody Plants. St. Paul: APS    Press. pp. 87-100.-   Read, D. J.: 1999, ‘Mycorrhiza—the state of the art’, in A. Varma    and B. Hock (eds.), Mycorrhiza, Berlin, Springer-Verlag, pp. 3-34.-   Redman R S, Freeman S, Clifton D R, Morrel J, Brown G, Rodriguez R J    (1999). Biochemical analysis of plant protection afforded by a    nonpathogenic endophytic mutant of colletotrichum magna. Plant    Physiology 119: 795-804.-   Redman R S, Dunigan D D, Rodriguez R J (2001). Fungal symbiosis:    from mutualism to parasitism, who controls the outcome, host or    invader? New Phytologist 151: 705-716.-   Redman R S, Rossinck M R, Maher S, Andrews Q C, Schneider W L,    Rodriguez R J (2002a). Field performance of cucurbit and tomato    plants infected with a nonpathogenic mutant of Colletotrichum magna    (teleomorph: Glomerella magna; Jenkins and Winstead). Symbiosis 32:    55-70.-   Redman R S, Sheehan K B, Stout R G, Rodriguez R J, Henson J M    (2002b) Thermotolerance generated by plant/fungal symbiosis. Science    298:1581.-   Redman R S, Kim Y O, Woodward C J, Greer C, Espino L, Doty S L,    Rodriguez R J (2011) Increased fitness of rice plants to abiotic    stress via habitat adapted symbiosis: a strategy for mitigating    impacts of climate change. PLoS One 6:e14823.-   Rodriguez R J, Yoder O C (1991). A family of conserved repetitive    DNA elements from the fungal plant pathogen Glomerella cingulata    (Colletotrichum lindemuthianum). Experimental Mycology 15: 232-242.-   Rodriguez R J (1993). Polyphosphates present in DNA preparations    from filamentous fungal species of Colletotrichum inhibits    restriction endonucleases and other enzymes. 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Other Embodiments

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

1.-18. (canceled)
 19. A method for increasing stress tolerance, themethod comprising: manually or mechanically inoculating a plant or aplant seed with an isolated Trichoderma harzianum fungus or a sporethereof; or a composition comprising the isolated Trichoderma harzianumfungus or a spore thereof, thereby increasing stress tolerance of theinoculated plant.
 20. The method of 19, wherein the stress is drought,elevated salt, reduced nutrients and/or temperature stress and/or thestress is not the presence of polycyclic aromatic hydrocarbons,napthenic acids, or high pH.
 21. (canceled)
 22. The method of claim 19,wherein inoculating the plant comprises colonizing a root, stem and/orleaf of the plant with a Trichoderma harzianum strain fungus or a sporeor progeny thereof.
 23. The method of claim 19, wherein the methodfurther comprises growing the plant or plant seed.
 24. The method ofclaim 23, wherein the plant or plant seed is grown in soil characterizedby high salinity, low moisture, and/or low nutrient content.
 25. Themethod of claim 24, wherein the high salinity is greater than 35 mMamount of a salt; the low moisture content is between 0-0.18 in³water/in³ soil; the low nutrient content is fewer than 80 lbs/acre ofnutrient, wherein the nutrient comprises 20% each of nitrogen,phosphorus, and potassium, and associated micronutrients; the plant orplant seed is grown in water characterized by high salinity and/or lownutrient content; and/or the plant or plant seed is grown or germinatedat an average temperature at or above 35 degrees Celsius or at or below15 degrees Celsius. 26.-29. (canceled)
 30. The method of claim 19,wherein the plant or plant seed is a crop plant or crop plant seed. 31.The method of claim 30, wherein the crop plant or crop plant seed iswatermelon, tomato, corn, wheat, soybean, cucurbits, peppers, leafygreens, barley, cotton, beans, peas, tubers, berries, woody plants orrice, or a seed thereof.
 32. The method of claim 19, wherein the plantor plant seed is an ornamental, such as Rosaceae, Liliaceae, Azalea,Rhododendron, Poaceae or Chrysanthemum.
 33. The method of claim 19,wherein the Trichoderma harzianum strain fungus is not strain TSTh20-1or strain T-22.
 34. A method for increasing germination of seeds, themethod comprising: manually or mechanically inoculating a plant seedwith an isolated Trichoderma harzianum fungus or a spore thereof; or acomposition comprising the isolated Trichoderma harzianum fungus or aspore thereof, thereby increasing germination of the inoculated seeds.35. The method of claim 34, wherein the plant seed is previously,concurrently and/or subsequently treated with a fungicide and/orinsecticide.
 36. The method of claim 34, wherein the plant or plant seedis a crop plant or crop plant seed, optionally wherein the crop plant orcrop plant seed is watermelon, tomato, corn, wheat, soybean, cucurbits,peppers, leafy greens, barley, cotton, beans, peas, tubers, berries,woody plants or rice, or a seed thereof.
 37. (canceled)
 38. The methodof claim 34, wherein the plant or plant seed is an ornamental, such asRosaceae, Liliaceae, Azalea, Rhododendron, Poaceae or Chrysanthemum. 39.The method of claim 34, wherein the Trichoderma harzianum strain fungusis not strain TSTh20-1 or strain T-22.
 40. A method for reducingestablishment of fungi other than Trichoderma harzianum in a plant, themethod comprising: manually or mechanically inoculating a plant seed orseedling with an isolated Trichoderma harzianum fungus or a sporethereof; or a composition comprising the isolated Trichoderma harzianumfungus or a spore thereof, thereby reducing establishment of fungi otherthan Trichoderma harzianum in a plant growing from the inoculated seedor seedling.
 41. The method of claim 40, wherein the plant or plant seedis a crop plant or crop plant seed.
 42. The method of claim 41, whereinthe crop plant or crop plant seed is watermelon, tomato, corn, wheat,soybean, cucurbits, peppers, leafy greens, barley, cotton, beans, peas,tubers, berries, woody plants or rice, or a seed thereof.
 43. The methodof claim 40, wherein the plant or plant seed is an ornamental, such asRosaceae, Liliaceae, Azalea, Rhododendron, Poaceae or Chrysanthemum. 44.The method of claim 40, wherein the Trichoderma harzianum strain fungusis not strain TSTh20-1 or strain T-22.